Aldolase, aldolase mutant, and method and composition for producing tagatose by using same

ABSTRACT

There are provided aldolase, an aldolase mutant, a method for producing tagatose, and a composition for producing tagatose using the same. The technical feature of the present invention is environment-friendly due to the use of an enzyme acquired from microorganisms, requires only a simple process of enzyme-immobilization, uses a low-cost substrate compared with that of a conventional method for producing tagatose, and has a remarkably high yield, thereby greatly reducing production cost while maximizing production effect.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 14/908,469 filed on Jan. 28, 2016, which is a National Stageapplication of PCT/KR2014/006850 filed on Jul. 25, 2014, which claimspriority to, and the benefit, Korean Patent Application No.10-2013-0089588 filed on Jul. 29, 2013, Korean Patent Application No.10-2014-0001709 filed on Jan. 7, 2014, and Korean Patent Application No.10-2014-0093443 filed on Jul. 23, 2014 in the Korea IntellectualProperty Office, the entire contents of which are incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to aldolase, an aldolase mutant, and amethod for producing tagatose and a composition for producing tagatoseusing the same.

BACKGROUND ART

Generally, tagatose (D-tagatose) is a C₄ epimer of fructose (D-fructose)and has a sugar content of 92% relative to sugar but it is a low-caloriesweetener having a calorie of 1.5 kcal/g, which is about 30% of sugar.Additionally, tagatose is a non-caloric sweetener which is hardlymetabolized during the in-vivo absorption process. About 15% to 20% ofthe amount of tagatose ingested is absorbed into the body but thisabsorption is due to the decomposition by the microorganisms in thelarge intestine not by self-digestion capability in humans, and it thusdoes not affect the blood glucose levels. Accordingly, it is expected toprovide a blood glucose level-controlling effect to diabetic patients,and is known to provide foods for enteric microorganisms, and therebyhelps with excretion activity by microorganisms. Tagatose has thefunctional characteristics of not causing tooth decay and thus it is ahealthy sweetener to be safely included in chocolate, gums, bread,candies, and the like, which are favored by children, instead of sugarso that children can intake without worries and has been highlighted asa material which can contribute to the prevention of diseases due toexcess sugar intake. Additionally, tagatose has a boiling point of 134°C. and a pH of 2 to 7, and is thus highly stable against heat and pH.Therefore, tagatose is not readily destroyed, unlike most artificialsweeteners, but has physical and chemical properties most similar tothat of sugar, and when it is heated, the characteristic of browningreaction is ketose, which is similar to that of fructose, and thus hasan important characteristic as a sugar substitute.

For these reasons, tagatose has been highlighted as a food supplementand a diet sweetener in food industry, and there is a growing need forthe development of a method for efficiently producing tagatose. This isbecause tagatose is a rare sugar included in dairy products or a fewplants in a small amount and it cannot be synthesized by a chemicalmethod. At present, tagatose is being produced by isomerization ofgalactose via a bioconversion method using L-arabinose isomerase.However, the supply of galactose is unstable and its supply price variesgreatly depending on the change in the market of dairy products thusraising a difficulty in securing a steady and large amount of product.

Accordingly, to solve these problems, studies have actively focused ondeveloping a method of producing tagatose using an enzyme based onsubstrates such as glucose or fructose, which has a low price and can besteadily supplied.

Until now, a single enzyme reaction that can produce tagatose fromfructose by a single enzyme reaction is not known. In the case of asingle enzyme, conversion can hardly occur by the mechanisms of enzymesfor epimerization known so far and the production yield obtainedtherefrom is significantly low and is thus not sufficient for itsindustrialization.

Prior Art Patent Document Korean Patent Application No. 10-2001-0080711DISCLOSURE Technical Problem

The present invention was made based on the above-described necessities,and an object of the present invention is to provide an enzyme used in amethod for preparing tagatose with high yield using fructose as asubstrate.

Another object of the present invention is to provide a method ofpreparing tagatose with high yield using fructose as a substrate.

A further object of the present invention is to provide a compositionfor preparing tagatose with high yield using fructose as a substrate.

Technical Solution

In order to achieve the above objects, the present invention provides acomposition that can mediate epimerization at C₄ of a monosaccharideincluding fructose 1,6-diphosphate aldolase as an active ingredient.

In an exemplary embodiment of the present invention, the fructose1,6-diphosphate aldolase is preferably one of the enzymes represented bythe amino acid sequences of SEQ ID NOS: 1 to 4, but any fructose1,6-diphosphate aldolase that can achieve the objects of the presentinvention by one or more mutation in these amino acid sequences, such assubstitution, deletion, inversion, and translocation, or mediate theepimerization of the C₄ of a different monosaccharide can also belong tothe scope of the present invention.

Additionally, the present invention provides a method of epimerizing theC₄ of a monosaccharide by treating with fructose 1,6-diphosphatealdolase.

Additionally, the present invention provides a composition for producingtagatose including fructose 1,6-diphosphate aldolase as an activeingredient.

In an exemplary embodiment of the present invention, the the fructose1,6-diphosphate aldolase is preferably one of the enzymes represented bythe amino acid sequences of SEQ ID NOS: 1 to 4, but any fructose1,6-diphosphate aldolase that can achieve the objects of the presentinvention by one or more mutation in these amino acid sequences, such assubstitution, deletion, inversion, and translocation, or other fructose1,6-diphosphate aldolase capable of producing tagatose can also belongto the scope of the present invention.

Additionally, the present invention provides a method of producingtagatose from fructose including reacting fructose 6-phosphate by addingaldolase thereto.

Additionally, the present invention provides a composition includingtagatose produced by the production method of the present invention asan active ingredient.

Additionally, the present invention provides a food compositionincluding tagatose, which can be produced using tagatose 6-phosphateproduced by the method of the present invention, as an activeingredient.

In an exemplary embodiment of the present invention, the food ispreferably beverages, chocolate, gums, bread, candies, dairy products,animal products, and the like, but is not limited thereto.

The present invention provides proteins represented by amino acidsequences of SEQ ID NOS: 1, 2, 3, and 4 which possess the activity ofepimerization of fructose 6-phosphate.

Additionally, to achieve another object of the present invention, thepresent invention provides a gene encoding proteins having the activityof epimerization of fructose 6-phosphate, represented by amino acidsequences of SEQ ID NOS: 1, 2, 3, and 4.

Additionally, to achieve still another object of the present invention,the present invention provides a recombinant expression vector includingthe above gene.

Furthermore, the present invention provides a method for producingtagatose by providing a method for producing tagatose 6-phosphate, whichis characterized by reacting a protein with fructose 6-phosphate.

Additionally, the present invention provides a composition for producingtagatose including a mutant of fructose 1,6-bisphosphate aldolase as anactive ingredient.

In an exemplary embodiment of the present invention, the fructose1,6-bisphosphate aldolase is preferably an enzyme selected from theenzymes consisting of the amino acids represented by SEQ ID NOS: 1 to 4,but is not limited thereto.

In another exemplary embodiment of the present invention, the mutant ispreferably a substitution one or more residues at positions of 332, 314,227, and 62 of fructose aldolased enzyme comprised of SEQ ID NO: 1,wherein the residue at position 332 is substituted from arginine toglutamine, the residue at position 314 is substituted from glutamine toalanine, the residue at position 227 is substituted from histidine toalanine, and the residue at position 62 is substituted from serine toalanine.

However, all mutants with an increased activity of the correspondingenzyme compared with that of the wild-type enzyme, by inducing amutation in other wild-type enzyme of fructose 1,6-bisphosphatealdolase, can belong to the protective scope of the present invention.For example, the mutant with an increased activity of the correspondingenzyme compared with that of the wild-type enzyme, by inducing amutation in any one of the enzymes represented by SEQ ID NOS: 2 to 4certainly belongs to the protective scope of the present invention.

In still another exemplary embodiment of the present invention, theabove composition is preferably further includes hexokinase and phytase,but is not limited thereto.

Additionally, the present invention provides a method for producingtagatose including treating a mutant of the fructose 1,6-bisphosphatealdolase of the present invention with fructose-6-phosphate.

In an exemplary embodiment of the present invention, thefructose-6-phosphate is preferably obtained by treating fructose or afructose-containing material with hexokinase, but when it is provided byother chemical syntheses it will also belong to the scope of the presentinvention.

In another exemplary embodiment of the present invention, the methodpreferably further includes converting the tagatose 6-phosphate intotagatose by acting phytase thereon, but the tagatose 6-phosphate may beremoved of its phosphate group by a different enzyme or chemical method.

Additionally, the present invention provides an enzyme for a mutantenzyme of fructose 1,6-bisphosphate aldolase, one of the enzymesselected from the enzymes consisting of the amino acids represented bySEQ ID NOS: 1 to 4.

Additionally, the present invention provides a gene encoding a mutant ofthe present invention.

In an exemplary embodiment of the present invention, hexokinase may berepresented by the amino acid sequence of SEQ ID NO: 5 or 6, but allcorresponding enzymes having the effect to be achieved in the presentinvention belong to the protective scope of the present invention.

According to the present invention, the productivity of tagatose wasincreased using the enzyme for epimerizing the C₄ of phosphate sugar,and the resolution of the problems, which occur when fermentation wasperformed using the producing method through the enzyme reaction of thecell itself, was prepared. In particular, there has been no precedentexample of producing tagatose from fructose, and the first suchproduction was attempted in the present invention. Additionally, when ayield close to 80% of tagatose can be obtained from fructose by acocktail reaction.

The present invention will be described in details herein below.

In particular, the technical terms and scientific terms as used herein,will refer to those which are commonly understood by a skilled person inthe art, unless defined otherwise.

Additionally, repeated explanations on the technical constitutions andactions equivalent to those of the conventional ones will be omittedherein below.

The characteristics of fructose 1,6-bisphosphate aldolase were confirmedby cloning a gene corresponding to the fructose 1,6-bisphosphatealdolased enzyme or protein derived from E. coli K-12, whosecharacteristics have not yet been confirmed in substrates other than thenatural substrate, i.e., fructose 1,6-bisphosphate, culturing amicroorganism transformed with an expression vector including the gene,followed by overexpression of the fructose 1,6-bisphosphate aldolase.

As a result, the present invention confirmed that the enzyme has thesubstrate specificity of epimerizing the C₄ of the fructose 6-phosphate,and thus the present invention relates to producing tagatose 6-phosphateusing the above enzyme and then treating with a commercial phytase tothereby produce tagatose.

More specifically, the gene for the known enzyme, fructose1,6-bisphosphate aldolase, is already known, but those bacteria, such asEscherichia coli K-12, Bacillus subtilis, Caldicellulosiruptorsaccharolyticus, and Kluyveromyces lactis, which have not been confirmedof their characteristics of epimerizing C₄ using fructose 6-phosphate,were used, and the present invention is the first in the world toconfirm that all these enzymes have the activity of converting fructose6-phosphate into tagatose 6-phosphate.

In particular, for the confirmation of the characteristics of theenzyme, the present invention preferably uses an enzyme obtained by:acquiring the gene for fructose 1,6-bisphosphate aldolase in a largeamount by polymerase chain reaction (PCR) from a bacterial strainincluding the gene for the known fructose 1,6-bisphosphate aldolase,which has been evaluated based on its nucleotide sequence by theprevious experiment and named accordingly without confirming thefunctional characteristics at all; inserting the gene into anappropriate expression vector to construct a recombinant vectorincluding the fructose 1.6-bisphosphate aldolase gene; culturing atransformed bacteria, which was prepared by transforming the recombinantvector into an appropriate microorganism, in a fermentation medium andoverexpressing the enzyme; and purifying.

Additionally, the method of producing tagatose of the present inventionconsists of three steps of obtaining fructose 6-phosphate by treatingfructose 1,6-bisphosphate aldolase, which was commonly called fructose1,6-bisphosphate aldolased enzyme, with hexokinase; obtaining tagatose6-phosphate by reacting the fructose 6-phosphate with a substrate; andobtaining tagatose by treating the tagatose 6-phosphate with phytase.

Additionally, the fructose 1,6-bisphosphate aldolased enzyme used inproducing tagatose of the present invention is not limited to the aminoacid sequences represented by SEQ ID NOS: 1 to 4, but any amino acidsequence as long as it can convert fructose 6-phosphate into tagatose6-phosphate, and in particular, even when there is substitution,insertion, deletion in a part of the amino acid sequences described inthese SEQ ID NOS.

Additionally, in the method of producing tagatose in the presentinvention, the expression vector to be used in cloning the gene forfructose 1,6-bisphosphate aldolase may be any vector including RSFDuet-1 that has been used in gene recombination, and as the bacterialstrain to be transformed with the recombinant vector, it is preferableto use E. coli BL21(DE3), but any bacterial strain, which can produce anactive protein via overexpression of a desired gene after beingtransformed with a gene recombinant vector, may be used

More specifically, regarding the cultivation of a microorganism in thepresent invention, E. coli BL21(DE3) [Escherichia coli BL21(DE3)] wasused as the recombinant bacterial strain to obtain fructose1,6-bisphosphate aldolase, and LB was used as a culture medium forproducing the microorganism, and a medium including 10 g/L of glycerol,1 g/L of peptone, 30 g/L of yeast extract, 0.14 g/L potassiumdiphosphate, and 1 g/L of monosodium phosphate was used as the mediumfor producing the enzyme. For the large-scale production of fructose1,6-bisphosphate aldolase, it is preferable to inoculate thefrozen-stored strain BL21(DE3) into a 250 mL flask including 50 mL of anLB medium, culture the bacterial strain in a shaking water bath at 37°C. until the absorbance at 600 nm reaches 2.0, add the culture into a 7L fermenter (Biotron, Korea) including a 5L of a fermentation medium andculture until the absorbance at 600 nm reaches 2.0, add with 1 mM IPTGto induce the production of the overexpressing enzyme, while maintainingthe stirring speed at 500 rpm, aeration of 1.0 vvm, and the culturetemperature at 37° C., during the process.

Additionally, for the purification of the fructose 1,6-bisphosphatealdolase produced by overexpression, the purified enzyme of the presentinvention is preferably obtained by the following process ofcentrifuging the culture of the transformed bacterial strain at 6,000×gat 4° C. for 30 minutes; washing the resulting cells twice with 0.85%NaCl, adding the cells in a cell lysate buffer solution (50 mM NaH₂PO₄,300 mM NaCl, pH 8.0) including 1 mg/mL of lysozyme, and placing in icefor 30 minutes; and crushing the cells in the solution by a French pressat 15,000 lb/in² and removing the cell lysate by centrifugation at13,000×g at 4° C. for 20 minutes, while purifying the supernatant byfiltering through a 0.45 μm filter paper. In particular, thepurification process is performed in a low-temperature room via fastprotein liquid chromatography (FPLC), in which the filtrate is appliedto a HisTrap HP column equilibrated with a 50 mM Tris-HCl buffersolution including 300 mM NaCl (pH 8.0) and 10 mM imidazole, andpreferably, the enzyme attached to the column after washing the columnwith the same buffer solution is eluted by flowing a solution, whichincludes imidazole at a concentration with a gradient from 10 mM to 200mM, at a rate of 1 mL/min Preferably, a fraction of the thus-elutedenzyme with an activity is added into a HiPrep 16/60 desalting resincolumn equilibrated with a 50 mM Tris-HCl buffer solution (pH 8.5), theadded protein is washed at a rate of 6 mL/min, the accumulated enzymesolution is added into a Sephacryl S-100 HR column equilibrated with 50mM Tris-HCl buffer solution including 0.15 M sodium chloride (pH 8.5) toelute the accumulated enzyme at a rate of 6.6 mL/min, and the elutedsolution is finally dialyzed in a 50 mM Tris-HCl buffer solution to beused.

Additionally, the thus-obtained fructose 1,6-bisphosphate aldolaseaccording to the present invention is a monomer having a molecularweight of 78 kDa, and is a metalloenzyme whose activation is controlledby metal ions.

In particular, it is preferable that the fructose 1,6-bisphosphatealdolase and fructose 6-phosphate are reacted at a ratio of 55% to 75%(w/w) at 50° C. (pH 8.5) considering the production yield of tagatose6-phosphate. This is because the production yield of tagatose6-phosphate was excellent when the concentration of the substrate forfructose was in the range from 55% to 75% (w/w), and the above pH andthe temperature were the optimum conditions for the fructose1,6-bisphosphate aldolased enzyme.

Meanwhile, the cocktail reaction of the present invention is a methodfor producing tagatose in a large-scale by reacting fructose within acell using hexokinase, fructose 1,6-bisphosphate aldolased enzyme, andphytase, and it is preferable to use kinase and fructose1,6-bisphosphate aldolase expressed in a large amount by overexpressingthem within a cell. This is because the intracellular environmentenables to maintain the activity of an overexpressed enzyme for a longperiod of time and regenerate cofactors necessary for reactions, and theE. coli that can be used in the present invention may be any E. coli aslong as it can overexpress enzymes.

Since the method of producing tagatose according to the presentinvention uses enzymes obtained from microorganisms it isenvironment-friendly, requires only a simple enzyme-fixing process, andcan convert the production of tagatose from fructose in a method whichhas not been done previously, greatly reduce production cost, andmaximize the production effect.

Additionally, the tagatose produced in a large-scale method as describedabove may be effectively used by being added into functional foods andpharmaceutical drugs.

Hereinafter, the present invention will be explained in more details bythe exemplary embodiments. However, it should be obvious to a skilledperson in the art that the exemplary embodiments disclosed herein shouldnot be construed as limiting the scope of the present invention butcovering various alternatives and modifications as well as the exemplaryembodiments within the ideas and scope of the present invention.Accordingly, the appended claims should be appropriately interpreted tocomply with the spirit and scope of the present invention.

EFFECT

As described above, the characterization of novel enzymes according tothe present invention can provide an advantage in that the enzymes canbe used after selection to be suitable for various productionenvironment based on the similarities in characteristics between enzymesand the identity and conversion rate possessed by each enzyme.

Additionally, the method of producing tagatose of the present inventionis environment-friendly because only the enzymes obtained frommicroorganism are used, requires only a simply enzyme-fixing process,and compared with the conventional methods of producing tagatoseproduction, the method of the present invention uses only low-costsubstrates while having a significantly higher yield, and it thus canmarkedly reduce production cost and maximize production effect.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram illustrating the production of tagatosefrom fructose by a cocktail reaction introduced in the presentinvention.

FIGS. 2 to 4 illustrate the comparison results of phylogenetic trees andamino acid sequences with fructose 1,6-diphosphate aldolased enzymederived from Escherichia coli K-12, regarding the selection of fructose1,6-diphosphate aldolase of Streptococcus thermophilus,Caldicellulosiruptor saccharolyticus, and Kluyveromyceslactis introducedin the present invention.

FIG. 5 is a graph illustrating the relative activities of fructose1,6-diphosphate aldolased enzyme derived from Escherichia coli K-12 andfructose 1,6-diphosphate aldolased enzyme of fructose 1,6-diphosphatealdolase of Streptococcus thermophilus, Caldicellulosiruptorsaccharolyticus, and Kluyveromyceslactis after reacting with 5 mMfructose 6-phosphate at pH 8.5 at 50° C. for 1 hour.

FIG. 6 is a graph illustrating comparison results of the metalspecificity of fructose 1,6-diphosphate aldolases of the presentinvention.

FIGS. 7 to 9 are graphs illustrating the relative activity andconversion (FIG. 9) of tagatose 6-phosphate in fructose 6-phosphateaccording to an enzyme optimum pH (FIG. 7) and optimum temperature (FIG.8) for fructose 1,6-diphosphate aldolase of Streptococcus thermophilus,respectively.

FIGS. 10 to 12 are graphs illustrating the relative activity andconversion (FIG. 12) of tagatose 6-phosphate in fructose 6-phosphateaccording to an enzyme optimum pH (FIG. 10) and optimum temperature(FIG. 11) for fructose 1,6-diphosphate aldolase of Caldicellulosiruptorsaccharolyticus, respectively.

FIGS. 13 to 15 are graphs illustrating the relative activity andconversion (FIG. 15) of tagatose 6-phosphate in fructose 6-phosphateaccording to an enzyme optimum pH (FIG. 13) and optimum temperature(FIG. 14) for fructose 1,6-diphosphate aldolase of Kluyveromyces lactis,respectively.

FIG. 16 is a graph illustrating the results of the conversion offructose 6-phosphate into tagatose 6-phosphate using each of thefructose 1,6-diphosphate aldolases used in the present invention. Thefructose 1,6-diphosphate aldolase of Kluyveromyces lactis exhibited anactivity similar to that of the enzyme derived from Escherichia coliK-12, and the enzyme derived from Streptococcus thermophilus exhibited afast initial conversion but exhibited a slower conversion of 71%. Theenzyme derived from Caldicellulosiruptor saccharolyticus exhibited aconversion of about 80%, similar to that of the enzyme derived fromEscherichia coli K-12, and a fast initial conversion.

FIG. 17 is a schematic diagram of RSF Duet-1 vector system used in thepresent invention, and this system aims at cloning other enzymes alongwith fructose 1,6-diphosphate aldolase.

FIG. 18 is a graph illustrating the production activity obtained byreacting hexokinase derived from Saccharomyces cerevisiae with fructosein a concentration from 5 mM to 50 mM for 1 hour.

FIGS. 19 to 22 illustrate the metal specificity of fructose1,6-diphosphate aldolase of Escherichia coli of the present invention(FIG. 19), the conversion from fructose 6-phosphate into tagatose6-phosphate according to an optimum pH for fructose 1,6-diphosphatealdolase of Escherichia coli (FIG. 20), the conversion according to anoptimum temperature (FIG. 21), respectively. FIG. 22 is a graphillustrating the result of converting 10 mM fructose 6-phosphate intotagatose 6-phosphate under the optimum conditions confirmed in theresults of FIGS. 19 to 21.

FIG. 23 lists the conversion into tagatose according to theconcentration of phytase when reacting the tagatose 6-phosphate, whichwas converted in the present invention, with phytase.

FIG. 24 is a graph illustrating the comparison result of enzyme activitybetween gene-mutating enzymes constructed in the present invention and awild-type enzyme, in which increased conversion rate and improvedproductivity are obtained by a faster conversion rate.

BEST MODE

Hereinafter, the present invention will be described in detail withreference to Examples. However, the scope of right of the presentinvention is not limited by these Examples.

EXAMPLE 1 Large-Scale Production of Fructose 1,6-diphosphate aldolasedEnzyme

Regarding fructose 1,6-diphosphate aldolase genes, DNA from Escherichiacoli strain K-12, and each strain of Streptococcus thermophilus,Caldicellulosiruptor saccharolyticus, and Kluyveromyces lactis weresuggested as genes for fructose 1,6-diphosphate aldolase, but they wereobtained in a large-scale by performing PCR amplification afterdesigning primers (see Table 2) based on the nucleotide sequence of DNAof genes, which have never been identified, inserting the PCR productinto an RSF Duet-1 vector [Novagen] using restriction enzymes, Sal I andNot I to construct a recombinant vector, RSF Duet-1/fructose1,6-diphosphate aldolase, followed by transforming the recombinantvector into E. coli BL21(DE3) by a conventional transformation method.Additionally, the E. coli BL21 strain was stored in liquid nitrogenprior to cultivation for a large-scale production.

Then, for a large-scale production of fructose 1,6-diphosphate aldolase,first, the frozen-stored BL21(DE3) strain was inoculated into a 250 mLflask including 50 mL of LB and seed-cultured in a shaking water bathmaintained at 37° C. until the absorbance at 600 nm reached 2.0, and theseed-cultured culture broth was subjected to a main cultivation byadding it into a 7 L fermentor (Biotron, Korea) including a 5 L of afermentation medium (10 g/L of glycerol, 1 g/L of peptone, 30 g/L ofyeast extract, 0.14 g/L potassium diphosphate, and 1 g/L of monosodiumphosphate). In particular, the large-scale production of fructose1,6-diphosphate aldolase was induced by adding 1 mM ITPG when theabsorbance at 600 nm reached 2.0. Specifically, the stirring speed at500 rpm, aeration of 1.0 vvm, and the culture temperature at 37° C. weremaintained during the above process.

EXAMPLE 2 Purification of Fructose 1,6-diphosphate aldolase

In order to accurately identify the characteristics of fructose1,6-diphosphate aldolase,

the enzyme was purified using affinity HisTrap HP column, desaltingHiPrep 16/60, and gel filtration Sephacryl S-100 HR column.

EXAMPLE 3 Metal Specificity of fructose 1,6-diphosphate aldolase

According to previous reports, fructose 1,6-diphosphate aldolase isinvolved in the conversion of 1,6-diphosphate substrate intodihydroxyacetone phosphate and glyceraldehyde 3-phosphate by metal zincand improve titer. However, the present invention confirmed that a metalsalt effect does not affect to increase titer when fructose 6-phosphatewas applied as a substrate. In order to examine the metal salt effect,the enzyme activity was measured after treating with EDTA or adding 1 mMmetal ions, as illustrated in figures below, and in particular, thereaction was performed in a 50 mM PIPES buffer solution (pH 8.5)including 0.15% fructose and 0.05 U/mL at 50° C. for 30 minutes, and theenzyme activity was measured after stopping the reaction with 0.2 M HCl.

As a result, it was confirmed that the fructose 1,6-diphosphate aldolaseof the present invention exhibited no change in its activity by metalions, and unlike as disclosed in previous reports, zinc ions wereexhibited to be a metal enzyme that can significantly inhibit enzymeactivity.

EXAMPLE 4 Activity of fructose 1,6-diphosphate aldolase According toChanges in pH and Temperature

In the present Example, in order to examine the activity of fructose1,6-diphosphate aldolase according to changes in pH and temperature, theenzyme and the substrate were reacted at various pH and temperatures tocompare the enzyme activity. In particular, to examine the effect of pH,the reaction was performed in a 50 mM Trizma base buffer solutionincluding 0.15% fructose 6-phosphate and 0.05 U/mL of the enzyme at a pHfrom 7.0 to 9.0. Specifically, the reaction was performed at 50° C. for1 hour. Then, 0.2 M HCl was added to stop the reaction and the enzymeactivity was measured. The results are illustrated in each figure.

Additionally, in order to examine the effect of temperature, thereaction was performed in a 50 mM Trizma base buffer solution (pH 8.5)including 0.15% fructose 6-phosphate and 0.05 U/mL of the enzyme at atemperature from 30° C. to 70° C. for 1 hour. Specifically, 0.2 M HClwas added to stop the reaction and the enzyme activity was measured. Theresults are illustrated in each figure. As a result, the optimum pH wasexhibited to be 8.5, being similar in both Streptococcus thermophilusand Kluyveromyceslactis, and their activities were exhibited to beindependent of pH. The optimum temperature for each of the enzymes wasexhibited to be 50° C., and Streptococcus thermophiles also showed 91%of relative activity at 30° C.

Based on the above results, it was confirmed that the conversion offructose 6-phosphate into tagatose 6-phosphate at optimum temperatureand pH according to time zone could reach from 70% to 80%, and theresults are illustrated in figures. However, regarding the abovereaction, any reaction in any range according to the desired yield orreaction conditions may be applied without defining particular pH ortemperature.

EXAMPLE 5 Activity of Conversion from Fructose to fructose 6-phosphateby Hexokinase

For the production of tagatose at high concentration, as the first step,to produce fructose 6-phosphate by reacting fructose at a concentrationof from 5 mM to 50 mM with an equal amount of adenosine triphosphate(ATP) and hexokinase derived from Saccharomyces cerevisiae, reacted with250 U/mL of the enzyme included in a 50 mM Tris buffer solution (pH 7.5)at 30° C. for 60 minutes. Then, the enzyme activity was measured. Theamount of fructose 6-phosphate production according to enzymeconcentration is illustrated in FIG. 18. As a result, fructose6-phosphate at a concentration of from 5 mM to 50 mM was produced, andthis corresponds to 90% or higher of conversion.

The hexokinase used in this Example was lyophilized powder, H4502 TypeF-300 purchased from Sigma Aldrich (130 U/mg protein (biuret), Sigma)and the phytase was Genophos 10000G purchased from Genofocus, Inc.

EXAMPLE 6 Large-Scale Production of fructose 1,6-bisphosphate aldolasedEnzyme

Fructose 1,6-diphosphate aldolase gene was obtained in a large-scale byperforming PCR amplification after designing primers based on thenucleotide sequence of DNA of Escherichia coli strain K-12 substrainMG1655, inserting the PCR product into an RSF Duet-1 vector [Novagen]using restriction enzymes, Sal I and Not I to construct a recombinantvector, RSF Duet-1/fructose 1,6-diphosphate aldolase (FIG. 17), followedby transforming the recombinant vector into E. coli BL21(DE3) by aconventional transformation method. Additionally, the recombinant E.coli strain was stored in liquid nitrogen prior to cultivation for alarge-scale production.

For a large-scale production of fructose 1,6-diphosphate aldolase, thefrozen-stored BL21(DE3) strain was inoculated into a 250 mL flaskincluding 50 mL of LB and seed-cultured in a shaking water bathmaintained at 37° C. until the absorbance at 600 nm reached 2.0, and theseed-cultured culture broth was subjected to a main cultivation byadding it into a 7 L fermentor (Biotron, Korea) including a 5L of afermentation medium (10 g/L of glycerol, 1 g/L of peptone, 30 g/L ofyeast extract, 0.14 g/L potassium diphosphate, and 1 g/L of monosodiumphosphate). In particular, the large-scale production of fructose1,6-diphosphate aldolase was induced by adding 1 mM ITPG when theabsorbance at 600 nm reached 2.0. Specifically, the stirring speed at500 rpm, aeration of 1.0 vvm, and the culture temperature at 37° C. weremaintained during the above process.

EXAMPLE 7 Production of Tagatose from tagatose 6-phosphate using Phytase

For the production of tagatose at high concentration, 10 mM tagatose6-phosphate converted from fructose 6-phosphate was reacted with 10 to50 U/mL of phytase in a 50 mM pH 7.5 Trizma buffer solution (pH 5.5) at60° C. for 60 minutes. Then, the enzyme activity was measured. Theamount of tagatose production according to enzyme concentration islisted in FIG. 23.

As a result, 9 mM of tagatose was produced for 50 U/mL of cultivationtime, and this corresponds to 90% of conversion yield.

Example 8 Production of Tagatose from Fructose by a Cocktail Reaction ofHexokinase, Aldolase, and Phytase

Tagatose was produced from fructose by a cocktail reaction ofhexokinase, aldolase, and phytase based on the Examples above. Fructose6-phosphate was produced by reacting 5 mM fructose with an equal amountof adenosine triphosphate (ATP) and 250 U/mL of hexokinase derived fromSaccharomyces cerevisiae in a 50 mM Trizma buffer solution (pH 7.5) at30° C. for 60 minutes, and as a result, 100% of the 5 mM fructose wasconverted into 5 mM fructose 6-phosphate. As a serial reaction, when a50 mM Trizma base buffer solution including 0.5 U/mL of fructose1,6-bisphosphate aldolase was reacted at pH 8.5 for 30 minutes, 93% ofthe 5 mM fructose 6-phosphate was converted into 4.65 mM tagatose6-phosphate. Then, when the reaction was performed in a 50 mM Trizmabase buffer solution (pH 5.5) including 50 U/mL of the enzyme at 60° C.for 60 minutes, 100% of the 4.65 mM tagatose 6-phosphate was convertedinto 4.65 mM tagatose. Conclusively, as a result of the cocktailreaction of hexokinase, aldolase, and phytase using 5 mM fructose, 93%was successfully converted into 4.65 mM tagatose.

EXAMPLE 9 Change in Activity of Gene Mutant Enzyme According to AminoAcid Substitution of Aldolase

For the production of tagatose at high concentration of the presentinvention, in order to increase the activity of aldolase, an amino acidsubstitution was caused by manipulating basic gene sequence and thechange in activity of the enzyme was observed. As a result, a genemutant enzyme, which can exhibit a fast conversion effect through afaster initial reaction speed, was successfully constructed. The genesequences encoding the amino acids to be mutated were mutated with sitedirected mutation and thereby a gene mutant enzyme was constructed. Sitedirected mutation was performed using the Muta-Direct™ Site DirectedMutagenesis Kit, and primers, in which the genes encoding 332R, 314Q,227H, and 62S, i.e., the amino acids to be mutated, were substituted toencode glutamic acid or alanine (see sequences in Table 1), wereconstructed to amplify a recombinant plasmid, which was sequenced aftertransformation, and the strains having substituted mutant enzymes wereselected via screening. The selected gene mutant enzymes were subjectedto purification in the same manner as in wild-type strain according toExample 2, and reacted in a 50 mM Trizma base buffer solution (pH 8.5)including 1.0% fructose 6-phosphate and 0.04 U/mL of the enzyme for 10minutes, for comparison of activities. In particular, the reaction wasstopped by adding 0.2 M HCl, the amount of the converted tagatose6-phosphate and the fructose 6-phosphate was analyzed, the enzymeactivity was measured by converting the activity of the wild-type enzymeinto relative activity 100%, and the results are illustrated in FIG. 24.As a result, the R332Q mutant showed an increase of about 140%, theQ314A showed an increase of about 250%, the H227A mutant showed anincrease of about 230%, and the S62A mutant showed an increase of about150%, relative to that of the wild-type enzyme, respectively.

TABLE 1 Name Nucleotide sequence 5 to 3' S62AGGTTATCGTTCAGTTCGCCAACGGTGGTGCTTC (SEQ ID NO: 7) S62A antiGAAGCACCACCGTTGGCGAACTGAACGATAACC  (SEQ ID NO: 8) H227AGCGTCCTTCGGTAACGTAGCCGGTGTTTACAAG (SEQ ID NO: 9) H227A antiCTTGTAAACACCGGCTACGTTACCGAAGGACGC  (SEQ ID NO: 10) Q314ACTTATCTGCAGGGTGCGCTGGGTAACC (SEQ ID NO: 11) Q314A antiGGTTACCCAGCGCACCCTGCAGATAAG (SEQ ID NO: 12) R331QTACGATCCGCAGGTATGGCTGCGTGCCG (SEQ ID NO: 13) R331Q antiCGGCACGCAGCCATACCTGCGGATCGTA (SEQ ID NO: 14)

Table 1 lists information on primers used in constructing mutants offructose 1,6-bisphosphate aldolase.

TABLE 2 Escherichia coli Sal I GTCGAC TCTAAGATTTTTGATTTCGTAAAACC (SEQ(strain K12) ID NO: 15) Not I GCGGCCGC TTACAGAACGTCGATCGCGTT (SEQ IDNO: 16) Streptococcus Sal I GTCGAC   GCAATCGTTTCAGCAGAAAAATTTG (SEQthermophilus ID NO: 17) Not I GCGGCCGC  TTAAGCTTTGTTTGCTGAACC (SEQ IDNO: 18) Caldicellulosiruptor Sal I GTCGAC  CCACTTGTAACAACCAAAGAG (SEQ ID saccharolyticus NO: 19) Not I GCGGCCGCTTAGCCTCTGTTCTTCTTAATCTC (SEQ ID NO: 20) Kluyveromyces Sal I GTCGAC CCAGCTCAAGACGTATTGACCAG (SEQ ID lactis NO: 21) No tI GCGGCCGC TTATTCCAAAGCACCCTTAGTAC (SEQ ID NO: 22)

Table 2 lists information on primers used in the present invention foreach of fructose 1,6-diphosphate aldolase gene.

1. A method of producing tagatose from fructose, the method comprising:reacting fructose 6-phosphate with tagatose 6-phosphate epimerase or amutant thereof to obtain tagatose 6-phosphate; and converting thetagatose 6-phosphate to tagatose.
 2. The method of claim 1, wherein thetagatose 6-phosphate epimerase catalyzes the conversion of fructose6-phosphate to tagatose-6-phosphate.
 3. The method of claim 1, whereinthe tagatose 6-phosphate epimerase is SEQ ID NOS:
 1. 4. The method ofclaim 1, wherein the tagatose 6-phosphate epimerase is SEQ ID NOS:
 2. 5.The method of claim 1, wherein the tagatose 6-phosphate epimerase is SEQID NOS:
 3. 6. The method of claim 1, wherein the tagatose 6-phosphateepimerase is SEQ ID NOS:
 4. 7. The method of claim 1, wherein the mutantcomprises at least one amino acid substitution of SEQ ID NO: 1 selectedfrom the group consisting of: i) a substitution of arginine residue withglutamine at a position corresponding to position 332, ii) asubstitution of glutamine residue with alanine at a positioncorresponding to position 314, iii) a substitution of histidine residuewith alanine at a position corresponding to position 227, and iv) asubstitution of serine residue with alanine at a position correspondingto position
 62. 8. The method of claim 1, wherein the fructose6-phosphate is obtained by treating fructose or a fructose-containingmaterial with hexokinase.
 9. The method of claim 1, wherein the tagatoseis obtained by treating the tagatose 6-phosphate with phytase.
 10. Themethod of claim 1, wherein the reaction of the fructose 6-phosphate withthe tagatose 6-phosphate epimerase is performed at a pH from 7.0 to 9.0.11. The method of claim 1, wherein the reaction of the fructose6-phosphate with the tagatose 6-phosphate epimerase is performed at atemperature from 30 ° C. to 70 ° C.